Enzymatic measurement of phosphatidic acid in cultured cells

Shin-ya Morita; Kazumitsu Ueda; Shuji Kitagawa

doi:10.1194/jlr.D900014-JLR200

Enzymatic measurement of phosphatidic acid in cultured cells

^*Laboratory of Pharmaceutical Technology, Kobe Pharmaceutical University, Higashinada-ku, Kobe 658-8558, Japan
^†Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
^§Institute for Integrated Cell-Material Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

1To whom correspondence should be addressed. e-mail: smorita{at}kobepharma-u.ac.jp

Abstract

In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes.

Footnotes

Abbreviations:

DAOS

N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline

DGK

diacylglycerol kinase

G3P

glycerol-3-phosphate

GPO

glycerol-3-phosphate oxidase

LPA

lysophosphatidic acid

MGL

monoglyceride lipase

mTOR

mammalian target of rapamycin

PA

phosphatidic acid

PC

phosphatidylcholine

PE

phosphatidylethanolamine

PI

phosphatidylinositol

PS

phosphatidylserine
This work was supported in part by Japan Heart Foundation Young Investigator’s Research Grant and by a grant from Hyogo Science and Technology Association.